1Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany.

Abstract

Listeria monocytogenes is a gram-positive facultative intracellular pathogen, causing serious illness in immunocompromised individuals and pregnant women. Upon detection by macrophages, which are key players of the innate immune response against infection, L. monocytogenes induces specific host cell responses which need to be tightly controlled at transcriptional and post-transcriptional levels. Here, we ask whether and how host miRNAs, which represent an important mechanism of post-transcriptional regulation in a wide array of biological processes, are altered by a model pathogen upon live infection of murine bone marrow derived macrophages. We first report that L. monocytogenes subverts the host genome-wide miRNA profile of macrophages in vitro. Specifically, we show that miR-155, miR-146a, miR-125a-3p/5p and miR-149 were amongst the most significantly regulated miRNAs in infected macrophages. Strikingly, these miRNAs were highly upregulated upon infection with the Listeriolysin-deficient L. monocytogenes mutant Δhly, that cannot escape from the phagosome thus representing a vacuolar-contained infection. The vacuolar miRNA response was significantly reduced in macrophages deficient for MyD88. In addition, miR-146a and miR-125a-3p/5p were regulated at transcriptional levels upon infection, and miR-125a-3p/5p were found to be TLR2 responsive. Furthermore, miR-155 transactivation in infection was regulated by NF-κB p65, while miR-146a and miR-125a-3p/5p expression was unaffected in p65-deficient primary macrophages upon L. monocytogenes infection. Our results demonstrate that L. monocytogenes promotes significant changes in the miRNA expression profile in macrophages, and reveal a vacuolar-dependent miRNA signature, listeriolysin-independent and MyD88-dependent. These miRNAs are predicted to target immune genes and are therefore most likely involved in regulation of the macrophage innate immune response against infection at post-transcriptional levels.

Bone marrow derived macrophages (BMDMs) were infected (MOI 10) with L. monocytogenes (Lm) and the LLO-deficient mutant Δhly (Lm Δhly) for 3 h and 6 h. Total RNA was extracted and the expression levels of primary transcript (pri)-miR-125a (A) and pri-miR-146a (B) were quantified by RT-qPCR using TaqMan assays. Data was normalized to the endogenous control gene HPRT1 and fold changes of miRNA induction in infected compared to control cells of each genotype were calculated by the 2−ΔΔCT method. Data represents the mean ± SEM from three biological replicates. Statistical significance of miRNA expression between infected and non infected WT BMDMs was determined by the Student t-test; * P<0.05, ** P<0.01, *** P<0.001. Statistical significance of miRNA induction in infection between WT and MyD88−/− BMDMs was determined by two-way ANOVA test; # P<0.05, ## P<0.01, ### P<0.001.

Regulation of miR-125a-3p/5p, miR-146a and miR-155 expression in macrophages by NF-κB p65.

Wild type (WT) and p65MYEL KO BMDMs were treated with 100 ng/ml ultra-pure LPS for 4 h and 8 h (A–D, F) or infected (MOI 1) with L. monocytogenes (Lm) for 4 h (E–F). Total RNA was extracted and the expression levels of miR-125a-3p, miR-125a-5p, miR-146a and miR-155 were quantified by TaqMan miRNA assays. Data was normalized to sno202 or HPRT1, endogenous controls for miR or pri-miR expression, respectively, and fold changes were calculated by the 2−ΔΔCT method and expressed relative to the mock-treated/mock-infected control for each timepoint/condition in each genotype. Data represents the mean ± SEM of at least two independent experiments, including minimum two mice from each genotype. Statistical significance of miR/pri-miR induction between WT and p65MYEL KO BMDMs was determined by two way ANOVA; * P<0.05, ** P<0.01, *** P<0.001.